PROJECT SUMMARY T cells expressing CD19-specific chimeric antigen receptors (CAR.CD19) can produce high rates of complete remission among patients with refractory B-cell malignancies. However, these studies have also revealed (i) difficulties in preparing sufficient numbers of CAR-T cells from the majority of pediatric patients with non-Hodgkin lymphoma (NHL), and (ii) a growing fraction of NHL patients with delayed relapse due to inadequate persistence of CAR-T cells or loss of CD19 from tumor cells. The long-term goal of Project 4 is to develop a safe and effective immunotherapy for NHL using both the natural and engineered properties of CD1d-restricted Va24-invariant natural killer T (NKT) cells. NKTs are attractive candidates for immunotherapy. They have antilymphoma activity via direct cytotoxicity against CD1d+ lymphoblasts or by activation of other immune effectors, such as NK cells; further, allogeneic NKTs do not produce graft- versus-host disease (GvHD) and can be prepared as ?off-the-shelf? products. We hypothesize that allogeneic NKTs engineered to express CAR.CD19 will show curative potential against NHL without the introduction of GvHD, a concept supported by our preliminary findings: NKTs transduced with CAR.CD19 directly kill CD19+ B lymphoblasts, can be expanded to clinical scale, and exert potent antitumor activity in xenogeneic lymphoma models. We have also shown that the CD62L+ subset of NKTs is essential for CAR.CD19-Tcell persistence and antitumor activity in vivo, and have devised means to preserve this subset of cells during CAR.CD19-NKT expansion. The following three specific aims will test our hypothesis: 1) Generate allogeneic CAR.CD19-NKTs with preserved CD62L expression and maximal antilymphoma potential, using the costimulatory aAPCs (artificial antigen-presenting cells, previously generated during the current funding period). 2) Determine the safety and antitumor activity of third-party CAR.CD19-NKTs in adult and pediatric patients with relapsed/refractory B-cell NHL. 3) Correlate the persistence, phenotype and function of CAR.CD19-NKTs with clinical responses. This study will the first in man to test the feasibility and therapeutic potential of immunotherapy with CAR-redirected NKTs. Our emphasis on CAR.CD19, with its favorable track record when expressed by T cells in NHL patients, will allow us to assess NKTs as a novel platform for NHL immunotherapy and perhaps other types of cancer as well.